Registration Dossier
Registration Dossier
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EC number: 203-940-1 | CAS number: 112-15-2
Toxicity to reproduction
Administrative data
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From October 08, 2020 to August 17, 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 021
- Report date:
- 2021
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- Adopted 29th July 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
Test material
- Reference substance name:
- 2-(2-ethoxyethoxy)ethyl acetate
- EC Number:
- 203-940-1
- EC Name:
- 2-(2-ethoxyethoxy)ethyl acetate
- Cas Number:
- 112-15-2
- Molecular formula:
- C8H16O4
- IUPAC Name:
- 2-(2-ethoxyethoxy)ethyl acetate
- Test material form:
- liquid
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source of test material: Production lot
- Lot/batch number of test material: P230220120
- Expiration date of the lot/batch: 2023-04-29
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Cool, well ventilated place
- Stability under storage conditions: Stable
- Stability under test conditions: Stable
Test animals
- Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- Wistar strain RccHan: WIST
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Animal breeding facility, Jai Research Foundation
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: (P) 13-14 weeks
- Weight at study initiation: (P) Males: 347-415 g; Females: 227-294 g
- Fasting period before study: No
- Housing: Premating - Groups of 2 or 3 rats/sex/cage. Mating period - Groups of 2 rats/cage (one male plus one female). Post mating - Female rats were caged individually.
- Diet: Teklad Global 14% protein rodent diet ad libitum
- Water: Reverse osmosis filtered drinking water ad libitum
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24 deg C
- Humidity (%): 65-68 %
- Air changes (per hr): 16-17
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 2020-10-16 To: 2020-12-31
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Minimum twice weekly
VEHICLE
- Justification for use and choice of vehicle: Water was used as the vehicle as the test item was readily miscible in that medium
- Concentration in vehicle: 0, 10, 50 and 100 mg/mL
- Amount of vehicle: Dose volume of 10 mL/kg body weight administered - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Active ingredient content and homogeneity of formulations were analysed once before initiation of treatment and twice during the treatment period. Three aliquots (from the upper, middle and lower layers) from each of the dose formulations and one aliquot from the vehicle control were sampled. Analysis was by GC/FID. On each analytical occasion, the mean concentration was determined and compared with the nominal value. The acceptance criteria were ± 10% deviation from the nominal value and %CV < 10.
- Duration of treatment / exposure:
- Males: Two weeks prior to mating, during the mating (two weeks) until the day before scheduled sacrifice, for a total of 35 days
Females: Two weeks prior to mating, the variable time to conception, the duration of pregnancy and fourteen days after delivery - Frequency of treatment:
- Daily
Doses / concentrationsopen allclose all
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Control
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 15
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Based on outcome of a preliminary dose-ranging screen
- Rationale for animal assignment: Random
Examinations
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations for mortality, morbidity and visible signs of reaction to treatment
BODY WEIGHT: Yes
- Time schedule for examinations: Males - Weekly. Females - Weekly during premating and mating periods; gestation days 0, 7, 14, 20 and 25; lactation days 0, 4, 7, 14 and day of termination
FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Males - Weekly during the pre-mating and post-mating periods. Females - Weekly during the pre-mating period; on days 0, 7, 14, and 20 of gestation; on days 0, 4, 7, and 14 of lactation.
WATER CONSUMPTION AND COMPOUND INTAKE: No - Oestrous cyclicity (parental animals):
- Oestrous cycle length and pattern were evaluated by vaginal smears of individual rats during the pre-treatment period of two weeks.
Vaginal smear was monitored daily from the beginning of the treatment period until evidence of mating.
Vaginal smear from each pregnant rat was also observed on the day of terminal sacrifice. - Sperm parameters (parental animals):
- Parameters examined in all P male parental generations:
All male animals - Testis and epididymis weight
Control and high-dose group males - Detailed testicular histopathological examination was conducted with special emphasis on stages of spermatogenesis and histopathology interstitial testicular cell structure. The evaluation included identification of potential treatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen. Examination of the intact epididymis included the caput, corpus, and cauda, which was accomplished by evaluation of a longitudinal section. The epididymis was evaluated for leukocyte infiltration, change in prevalence of cell types, aberrant cell types and phagocytosis of sperm. - Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups.
GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead
- Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: All surviving animals following 35 days of treatment
- Maternal animals: All surviving animals on lactation day 15
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cranial, thoracic, and abdominal viscera.
ORGAN WEIGHTS / TISSUE PRESERVATION
The following tissues were weighed and preserved: Brain, testes, epididymides, prostate, seminal vesicles with coagulating gland, LABC muscl complex, Cowper's glands, glans penis, ovaries, uterus including cervix, thyroid
HISTOPATHOLOGY
Detailed testicular histopathological examination was conducted with special emphasis on stages of spermatogenesis and histopathology of the interstitial testicular cell structure. The evaluation included identification of potential treatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen. Examination of the intact epididymis included the caput, corpus, and cauda, was accomplished by evaluation of a longitudinal section. The epididymis was evaluated for leukocyte infiltration, change in prevalence of cell types, aberrant cell types and phagocytosis of sperm.
Histopathological examination of the ovary was carried out to detect potential treatment-related effects such as qualitative depletion/increase of primordial, secondary, antral, graffian follicles population, persistence and increased/decreased corpus luteum, ovarian degeneration/atrophy and stromal cell proliferation. - Postmortem examinations (offspring):
- SACRIFICE
- The F1 offspring were sacrificed at 14 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cranial, thoracic, and abdominal viscera.
HISTOPATHOLOGY / ORGAN WEIGTHS
The thyroid gland from one male pup and one female pup/litter was weighed and preserved. - Statistics:
- Parametric data (body weight, body weight gain/change, food consumption, organ weight, and prenatal data etc.) was subjected to Shapiro-Wilk’s test for normality check wherever applicable, followed by Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. If the data did not meet the normality, data were transformed to check the normality again. If transformed data did not meet the normality check, the Kruskal-wallis/Mann Whitney test was performed to calculate significance. If the data did not meet the homogeneity of variance, statistical analysis was extended following the decision tree of Gad (Gad, S.C., 2007). Non-parametric data (mortality rate, pregnancy rate, foetal observations etc.) were analysed using Chi-square test.
Count data (foetal count, number of corpora lutea, number of implants, number of live foetuses, number of dead foetuses, number of pre-implantation loss, number of post-implantation loss, number of resorptions) were be subjected to non-parametric Kruskal-Wallis test.
AGD was normalised (the ratio of AGD to the cube root of body weight) and then subjected to statistical analysis. - Reproductive indices:
- Male and female fertility index
Pre-coital interval
Gestation index
Gestation length
Number of implants
Pre-natal/post-implantation loss
Post-natal loss
Live birth index - Offspring viability indices:
- Number of live pups on post partum days 0, 4 and 14 / survival index
Body weight of pups
Incidence of pup abnormalities
Results and discussion
Results: P0 (first parental generation)
General toxicity (P0)
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Mild salivation was observed in all 15 males and 12 of 15 female rats, approximately 3 to 5 minutes after dosing and persisted for approximately 20 to 25 minutes, in the 1000 mg/kg bw/day dose group. This finding was considered a response to the dose formulation and toxicologically insignificant, hence considered as non-adverse in nature. No clinical sign of toxicity was observed in male and female rats from any other treatment groups
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Body Weight
Males - A marginal decrease (-3.88% compared with controls) in the mean body weight of male rats belonging to the 1000 mg/kg/day group was observed at the end of the treatment period. The mean body weight of rats belonging to the 100 and 500 mg/kg/day dose groups was comparable with controls.
Females - A marginal decrease (-2.97% compared with controls) in mean body weight of female rats, belonging to the 1000 mg/kg/day dose group was observed during the gestation period. Mean body weight during the pre-mating and lactation periods was comparable with controls. The mean body weight of rats belonging to the 100 and 500 mg/kg/day dose groups was comparable with that of controls during pre-mating, gestation, and lactation periods.
Body Weight Gain
Males – A statistically significant decrease in mean body weight gain of male rats of the 1000 mg/kg/day dose group was observed during treatment days 29-35 and 1-35 when compared with that of the control group. Mean body weight gain of rats belonging to the 100 and 500 mg/kg/day dose groups was comparable with that of controls except for a statistically significant increase in mean body weight gain during treatment days 8-15 in the 500 mg/kg/day dose group. This increase in mean body weight gain is considered as incidental in the absence of dose dependency.
Females – A marginal decrease in mean body weight gain of female rats belonging to the 1000 mg/kg/day dose group was observed during the gestation period. The mean body weight gain of rats belonging to the 100 and 500 mg/kg/day dose groups was comparable with that of controls during the pre-mating, gestation, and lactation periods. During the pre-mating period, a statistically significant increase in the mean body weight gain of rats belonging to the 100 (during pre-mating days 1-15), 500 (during pre-mating days 1-8), and 1000 (during pre-mating days 1-8 and 1-15) mg/kg/day dose groups was observed when compared with that of controls. These increases in mean body weight gain of female rats are due to typically seen low values in the control group and considered as incidental in the absence of dose-dependency.
The marginal decrease in mean body weight and body weight gain in male and female (gestation period) rats are correlated with marginal decreased mean food consumption and considered as effects of the test item but non-adverse in nature. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Males – A marginal decrease in mean food consumption of rats belonging to the 1000 mg/kg/day dose group was observed during treatment days 29-35 and 1-35 along with statistical significance during treatment days 8-15. Mean food consumption of rats treated at 100 and 500 mg/kg/day was comparable with controls.
Females - During the pre-mating period, the mean food consumption of female rats was comparable with that of the control group. During the gestation period, a marginal decrease in mean food consumption of rats belonging to the 1000 mg/kg/day dose group was observed. A statistically significant decrease in mean food consumption of rats was observed during lactation days 4-7, 7-14 and 0-14 in the 1000 mg/kg/day dose group, during lactation days 7-14 and 0-14 in the 500 mg/kg/day dose group, and during lactation days 7-14 in the 100 mg/kg/day dose group. In the absence of other supporting findings in the mean body weight and body weight gain, these changes are considered incidental and non-adverse in nature.
The marginal decrease in the mean food consumption of male and female (gestation period) rats was considered as an effect of treatment but non-adverse in nature. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- Treatment did not cause histopathological alterations in any examined organ.
Atrophy of the testes and epididymides of single male individuals of the control and high dose groups was observed. This lesion was considered to be spontaneous or incidental in nature and not treatment-related. - Histopathological findings: neoplastic:
- not examined
- Other effects:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Thyroid Hormone Analysis
Serum T3 level of parental males females (LD15) was comparable with that of the control group.
A statistically significant decrease in serum T4 level of parental males was observed in the 500 and 1000 mg/kg/day dose groups when compared with that of the control group. Serum T4 levels of LD 15 female rats was comparable with controls.
Serum TSH level of parental males and LD 15 females were comparable with controls.
These changes are considered incidental and unrelated to treatment in the absence of other supporting findings in thyroid weight and serum TSH level as well as lack of dose dependency.
Reproductive function / performance (P0)
- Reproductive function: oestrous cycle:
- no effects observed
- Description (incidence and severity):
- No effect of treatment was observed on the mean oestrous cycle length and the mean number of oestrous cycles
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- no effects observed
- Description (incidence and severity):
- Male fertility index, female fertility index, gestation index, parturition index, percentage of pregnant rats and mating index of treated rats were comparable with that of controls. The duration of gestation and pre-coital interval was also comparable with that of controls. The mean number of implants was comparable with that of the control group
Effect levels (P0)
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- clinical signs
- mortality
- body weight and weight gain
- food consumption and compound intake
- organ weights and organ / body weight ratios
- gross pathology
- histopathology: non-neoplastic
- reproductive function (oestrous cycle)
- reproductive function (sperm measures)
- reproductive performance
Target system / organ toxicity (P0)
- Critical effects observed:
- no
Results: F1 generation
General toxicity (F1)
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No treatment-related clinical signs were observed in the pups.
- Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- mortality observed, non-treatment-related
- Description (incidence and severity):
- A statistically significant increase in mortality index of the female and composite of male and female pups belonging to the 1000 mg/kg/day dose group was observed during post-natal days 0-4 when compared with that of the control group. The mortality index of male pups belonging to the 1000 mg/kg/day dose group was also increased without statistical significance during post-natal days 0-4 when compared with that of the control group. These effects are attributed to losses in two litters before PND 4. Observed values were within the range of historical control data for male and female pups and near the higher range of historical control data for composite of male and female pups hence these effects were considered as incidental and unrelated to treatment.
The mortality index of male, female, and composite of male and female pups belonging to the 100 and 500 mg/kg/day dose groups was comparable with controls. - Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- The mean body weight and body weight gain of male, female and composite of male and female pups, belonging to the 100, 500, and 1000 mg/kg bw/day dose groups were comparable with that of the control group.
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Anogenital distance (AGD):
- no effects observed
- Description (incidence and severity):
- Anogenital distance of male and female pups belonging to the 100, 500 and 1000 mg/kg bw/day dose groups was comparable with that of the control group.
- Nipple retention in male pups:
- no effects observed
- Description (incidence and severity):
- None of the male pups belonging to either control or treated groups showed retention of nipples.
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- Thyroid weight (absolute and relative) of PND14 pups was unaffected by treatment.
A statistically significant decrease in absolute weight of the thyroid gland was noted in female pups of the 100 mg/kg bw/day dose group which was considered as unrelated to treatment due to the lack of the dose-dependency. - Gross pathological findings:
- no effects observed
- Histopathological findings:
- not examined
- Other effects:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Thyroid hormone analysis
Statistically, a significant decrease in serum T4 level of PND 4 pups was also observed in 1000 mg/kg/day dose group when compared with that of the control group.
A statistically significant increase in serum T3 level of PND 14 pups was observed at all dose levels when compared with that of the control group. Serum T4 level of PND 14 pups was comparable with that of the control group. Serum TSH level of PND 14 pups were comparable with that of the control group.
These changes are considered incidental and unrelated to treatment in the absence of other supporting findings in thyroid weight and serum TSH level as well as lack of dose dependency.
Developmental neurotoxicity (F1)
- Behaviour (functional findings):
- not examined
Developmental immunotoxicity (F1)
- Developmental immunotoxicity:
- not examined
Effect levels (F1)
open allclose all
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 500 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No effect
- Dose descriptor:
- LOAEL
- Generation:
- F1
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- mortality
Target system / organ toxicity (F1)
- Critical effects observed:
- no
Overall reproductive toxicity
- Reproductive effects observed:
- not specified
Any other information on results incl. tables
Body Weight (g) of Parental Males
Day |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
1 |
389.45 |
14.03 |
15 |
389.31 |
18.34 |
15 |
385.59 |
14.81 |
15 |
389.30 |
16.22 |
15 |
8 |
402.57 |
10.73 |
15 |
402.02 |
12.18 |
15 |
399.37 |
14.55 |
15 |
398.47 |
14.13 |
15 |
15 |
411.11 |
10.80 |
15 |
411.90 |
12.35 |
15 |
412.15 |
14.26 |
15 |
405.13 |
15.98 |
15 |
22 |
416.39 |
13.81 |
15 |
418.28 |
15.31 |
15 |
416.08 |
15.01 |
15 |
407.15 |
16.76 |
15 |
29 |
423.25 |
14.73 |
15 |
418.21 |
17.96 |
15 |
419.05 |
20.64 |
15 |
413.63 |
21.61 |
15 |
35 |
431.66 |
16.68 |
15 |
435.48 |
21.92 |
15 |
421.99 |
23.80 |
15 |
414.90 |
22.28 |
15 |
Key: N = Number of observations
SD = Standard deviation
Body Weight (g) of Parental Females
Pre-mating Period
Pre-mating Day |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
1 |
248.77 |
16.30 |
15 |
249.24 |
13.25 |
15 |
247.52 |
13.29 |
15 |
244.33 |
12.43 |
15 |
8 |
247.57 |
15.72 |
15 |
251.15 |
14.43 |
15 |
251.12 |
14.45 |
15 |
247.56 |
11.53 |
15 |
15 |
248.41 |
15.91 |
15 |
255.00 |
16.46 |
15 |
252.92 |
15.16 |
15 |
250.10 |
11.40 |
15 |
Gestation Period
Gestation Day |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
0 |
246.92 |
9.86 |
11 |
258.46 |
15.75 |
15 |
253.23 |
15.66 |
15 |
251.37 |
12.81 |
14 |
7 |
268.11 |
11.87 |
11 |
275.59 |
17.96 |
15 |
271.33 |
16.93 |
15 |
268.67 |
15.53 |
14 |
14 |
296.87 |
14.10 |
11 |
300.23 |
18.68 |
15 |
296.57 |
17.65 |
15 |
293.49 |
18.95 |
14 |
20 |
347.45 |
19.95 |
11 |
348.35 |
21.35 |
15 |
344.34 |
18.47 |
15 |
337.13 |
26.15 |
14 |
Lactation Period
Lactation Day |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
0 |
268.34 |
21.07 |
12 |
269.91 |
18.10 |
15 |
264.98 |
18.81 |
15 |
264.51 |
19.61 |
14 |
4 |
277.09 |
17.22 |
12 |
277.81 |
15.64 |
15 |
276.92 |
18.52 |
15 |
264.06 |
28.16 |
14 |
7 |
281.55 |
18.16 |
12 |
282.45 |
14.52 |
15 |
282.67 |
18.59 |
15 |
273.54 |
21.48 |
14 |
14 |
285.38 |
15.43 |
12 |
284.91 |
13.66 |
15 |
283.68 |
20.04 |
15 |
280.38 |
16.28 |
14 |
Key: N = Number of observations
SD = Standard deviation
Note: Non pregnant female rats were excluded from statistical analysis of gestation body weight.
Rat N° 61 (control) was assumed mated and delivered on GD 13, so gestation data was not considered for statistical analysis.
Body Weight Gain (g) of Parental Males
During Days |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
1-8 |
13.12 |
5.66 |
15 |
12.71 |
7.60 |
15 |
13.78 |
8.92 |
15 |
9.17 |
4.46 |
15 |
8-15 |
8.54 |
3.03 |
15 |
9.88 |
3.50 |
15 |
12.78# |
5.07 |
15 |
6.65 |
4.39 |
15 |
15-22 |
5.27 |
5.74 |
15 |
6.38 |
6.55 |
15 |
3.93 |
4.83 |
15 |
2.02 |
4.74 |
15 |
22-29 |
6.86 |
5.54 |
15 |
-0.07 |
16.62 |
15 |
2.97 |
8.75 |
15 |
6.48 |
6.50 |
15 |
29-35 |
8.41 |
4.13 |
15 |
17.27 |
18.55 |
15 |
2.95 |
11.99 |
15 |
1.27@ |
3.10 |
15 |
1-35 |
42.21 |
11.39 |
15 |
46.17 |
14.14 |
15 |
36.40 |
17.23 |
15 |
25.60# |
16.06 |
15 |
Key: N = Number of observations
SD = Standard deviation,
#= Significantly different from controls (5%),Dunnett's test
@ = Significantly different from controls (1%), Student's t-test
Body Weight Gain (g) of Parental Females
Pre-mating Period
Pre-mating Days |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
1-8 |
-1.20 |
4.93 |
15 |
1.91 |
4.66 |
15 |
3.60# |
3.28 |
15 |
3.23# |
3.83 |
15 |
8-15 |
0.83 |
4.92 |
15 |
3.85 |
6.29 |
15 |
1.80 |
5.68 |
15 |
2.54 |
3.65 |
15 |
1-15 |
-0.37 |
6.87 |
15 |
5.76# |
7.48 |
15 |
5.40 |
6.53 |
15 |
5.77# |
4.75 |
15 |
Gestation Period
Gestation Days |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
0-7 |
21.19 |
3.69 |
11 |
17.13 |
5.65 |
15 |
18.10 |
5.26 |
15 |
17.30 |
5.61 |
14 |
7-14 |
28.76 |
3.35 |
11 |
24.65 |
3.61 |
15 |
25.24 |
4.77 |
15 |
24.82 |
4.96 |
14 |
14-20 |
50.57 |
7.61 |
11 |
48.11 |
7.05 |
15 |
47.77 |
7.52 |
15 |
43.64 |
9.55 |
14 |
0-20 |
100.53 |
11.75 |
11 |
89.89 |
10.46 |
15 |
91.11 |
11.98 |
15 |
85.76 |
17.08 |
14 |
Lactation Period
Lactation Days |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
0-4 |
8.75 |
6.68 |
12 |
7.90 |
8.46 |
15 |
11.94 |
6.85 |
15 |
-0.44 |
24.87 |
14 |
4-7 |
4.46 |
6.28 |
12 |
4.64 |
5.36 |
15 |
5.75 |
6.47 |
15 |
9.47 |
25.26 |
14 |
7-14 |
3.83 |
7.29 |
12 |
2.46 |
6.74 |
15 |
1.01 |
6.32 |
15 |
6.84 |
10.11 |
14 |
0-14 |
17.04 |
13.54 |
12 |
15.00 |
10.50 |
15 |
18.70 |
13.00 |
15 |
15.87 |
11.97 |
14 |
Key: N= Number of observations
SD = Standard deviation
#= Significantly different from controls (5%),Dunnett's test
Note: Non pregnant female rats were excluded from statistical analysis of gestation body weight gain.
Rat N° 61 (control) was assumed mated and delivered on GD 13, so gestation data was not considered for statistical analysis.
Food Consumption (g/rat/day) of Parental Males
During Days |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
1-8 |
22.76 |
0.79 |
7 |
22.96 |
1.15 |
7 |
23.55 |
1.22 |
7 |
22.16 |
0.84 |
7 |
8-15 |
23.07 |
0.80 |
7 |
23.50 |
0.97 |
7 |
23.10 |
1.18 |
7 |
21.36# |
1.23 |
7 |
29-35 |
22.78 |
0.94 |
7 |
24.20 |
1.48 |
7 |
21.73 |
2.61 |
7 |
21.95 |
1.35 |
7 |
1-35 |
22.87 |
0.61 |
7 |
23.52 |
1.03 |
7 |
22.84 |
1.31 |
7 |
21.82 |
1.06 |
7 |
Key: N = Number of observations
SD = Standard deviation
# = Significantly different from controls (5%),Dunnett's test
Food Consumption (g/rat/day) of Parental Females
Pre-mating Period
Pre-mating Days |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
1-8 |
17.20 |
1.24 |
7 |
17.35 |
1.24 |
7 |
17.26 |
2.04 |
7 |
17.09 |
0.90 |
7 |
8-15 |
17.34 |
1.46 |
7 |
17.09 |
1.43 |
7 |
16.83 |
1.52 |
7 |
16.70 |
0.89 |
7 |
1-15 |
17.27 |
1.32 |
7 |
17.22 |
1.21 |
7 |
17.05 |
1.71 |
7 |
16.89 |
0.85 |
7 |
Gestation Period
Gestation Days |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
0-7 |
19.46 |
1.80 |
11 |
19.16 |
2.41 |
15 |
18.99 |
2.46 |
15 |
19.06 |
2.47 |
14 |
7-14 |
22.72 |
1.73 |
11 |
21.42 |
2.76 |
15 |
21.23 |
2.60 |
15 |
21.24 |
2.44 |
14 |
14-20 |
22.96 |
2.24 |
11 |
21.97 |
2.64 |
15 |
22.60 |
2.48 |
15 |
22.02 |
3.08 |
14 |
0-20 |
21.65 |
1.75 |
11 |
20.79 |
2.45 |
15 |
20.86 |
2.29 |
15 |
20.71 |
2.48 |
14 |
Lactation Period
Lactation Days |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
0-4 |
25.19 |
3.36 |
12 |
23.91 |
4.58 |
15 |
23.16 |
3.62 |
15 |
20.94 |
9.03 |
14 |
4-7 |
35.97 |
4.38 |
12 |
33.76 |
5.16 |
15 |
33.70 |
4.54 |
15 |
24.92**Φ |
11.47 |
14 |
7-14 |
45.03 |
1.98 |
12 |
43.27*@ |
2.36 |
15 |
41.93*Φ |
3.33 |
15 |
38.88**Φ |
5.95 |
14 |
0-14 |
37.42 |
2.09 |
12 |
35.70 |
3.19 |
15 |
34.80*@ |
3.19 |
15 |
30.76**Φ |
5.96 |
14 |
Key: N = Number of observations
SD = Standard deviation
* = Significantly different from controls (5%)
** = Significantly different from controls (1%)
Φ=Cochran's test
@ = Student's t-test
Note: Non pregnant female rats were excluded from statistical analysis of gestation food consumption.
Rat N° 61 (control) was assumed mated and delivered on GD 13, so gestation data was not considered for statistical analysis.
Number of Male Pups/Litter
Lactation Day |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
0 |
6.17 |
1.53 |
12 |
6.13 |
2.03 |
15 |
4.40^ |
1.76 |
15 |
5.77 |
1.42 |
13 |
4 |
5.83 |
1.27 |
12 |
5.53 |
1.92 |
15 |
4.13^ |
1.88 |
15 |
5.17 |
1.59 |
12 |
4R |
4.08 |
0.29 |
12 |
4.00 |
0.53 |
15 |
3.47 |
0.92 |
15 |
3.92 |
0.79 |
12 |
7 |
4.00 |
0.43 |
12 |
3.93 |
0.59 |
15 |
3.27 |
1.03 |
15 |
3.67 |
0.98 |
12 |
14 |
3.92 |
0.51 |
12 |
3.93 |
0.59 |
15 |
3.13 |
0.99 |
15 |
3.50 |
1.00 |
12 |
Key:N = Number of observations
SD = Standard deviation
R = Retained after standardization of litters
^= Significantly different from controls (5%),krushkal wallis test
Number of Female Pups/Litter
Lactation Day |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
0 |
6.33 |
1.78 |
12 |
5.13 |
1.30 |
15 |
6.27 |
2.37 |
15 |
4.79 |
2.52 |
14 |
4 |
6.00 |
1.76 |
12 |
5.00 |
1.41 |
15 |
5.93 |
2.05 |
15 |
4.08 |
2.33 |
13 |
4R |
3.92 |
0.29 |
12 |
4.00 |
0.53 |
15 |
4.20 |
1.08 |
15 |
3.08 |
1.19 |
13 |
7 |
3.83 |
0.39 |
12 |
3.93 |
0.59 |
15 |
4.13 |
0.99 |
15 |
3.00 |
1.15 |
13 |
14 |
3.75 |
0.45 |
12 |
3.93 |
0.59 |
15 |
4.07 |
1.10 |
15 |
3.00 |
1.04 |
12 |
Key:N = Number of observations
SD = Standard deviation
R = Retained after standardization of litters
Number of Male and Female Pups/Litter
Lactation Day |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
0 |
12.50 |
1.31 |
12 |
11.27 |
1.33 |
15 |
10.67 |
2.79 |
15 |
10.14 |
3.57 |
14 |
4 |
11.83 |
1.59 |
12 |
10.53 |
1.64 |
15 |
10.07 |
2.66 |
15 |
8.85 |
3.76 |
13 |
4R |
8.00 |
0.00 |
12 |
8.00 |
0.00 |
15 |
7.67 |
1.05 |
15 |
6.69^ |
2.10 |
13 |
7 |
7.83 |
0.39 |
12 |
7.87 |
0.35 |
15 |
7.40 |
1.30 |
15 |
6.38 |
2.14 |
13 |
14 |
7.67 |
0.65 |
12 |
7.87 |
0.35 |
15 |
7.20 |
1.37 |
15 |
6.00 |
2.20 |
13 |
Key:N = Number of observations
SD = Standard deviation
R = Retained after standardization of litters
^= Significantly different from controls (5%),krushkal wallis test
Male Sex Ratio (%)
Postnatal Day |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
0 |
49.54 |
11.65 |
12 |
53.59 |
13.88 |
15 |
42.02 |
13.94 |
15 |
52.33 |
19.93 |
14 |
Key:N = Number of observations
SD = Standard deviation
Body Weight (g) of Male Pups
Lactation Day |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
0 |
5.80 |
0.36 |
12 |
5.82 |
0.40 |
15 |
6.05 |
0.94 |
15 |
5.78 |
0.72 |
13 |
4 |
8.92 |
1.02 |
12 |
9.01 |
0.92 |
15 |
9.42 |
2.25 |
15 |
9.55 |
1.65 |
12 |
4R |
8.94 |
1.04 |
12 |
9.00 |
0.92 |
15 |
9.40 |
2.25 |
15 |
9.53 |
1.64 |
12 |
7 |
13.42 |
1.29 |
12 |
13.28 |
1.42 |
15 |
14.05 |
3.06 |
15 |
14.25 |
2.77 |
12 |
14 |
26.33 |
2.86 |
12 |
25.40 |
2.31 |
15 |
26.93 |
5.58 |
15 |
28.36 |
5.26 |
12 |
Key: N = Number of observations
SD = Standard deviation
R = Retained after standardization of litters
Body Weight (g) of Female Pups
Lactation Day |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
0 |
5.46 |
0.29 |
12 |
5.72 |
0.46 |
15 |
5.79 |
0.91 |
15 |
5.60 |
0.82 |
14 |
4 |
8.52 |
0.78 |
12 |
8.96 |
0.97 |
15 |
9.17 |
2.27 |
15 |
9.38 |
1.85 |
13 |
4R |
8.47 |
0.90 |
12 |
9.01 |
1.02 |
15 |
9.17 |
2.23 |
15 |
9.45 |
1.79 |
13 |
7 |
12.78 |
1.26 |
12 |
13.36 |
1.43 |
15 |
13.71 |
2.99 |
15 |
13.92 |
2.89 |
13 |
14 |
25.31 |
2.15 |
12 |
25.39 |
2.17 |
15 |
26.77 |
5.35 |
15 |
28.04 |
5.38 |
12 |
Key: N = Number of observations
SD = Standard deviation
R = Retained after standardization of litters
Body Weight (g) of Male and Female Pups
Lactation Day |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
0 |
5.63 |
0.31 |
12 |
5.77 |
0.42 |
15 |
5.89 |
0.92 |
15 |
5.72 |
0.74 |
14 |
4 |
8.72 |
0.88 |
12 |
8.98 |
0.95 |
15 |
9.26 |
2.27 |
15 |
9.48 |
1.66 |
13 |
4R |
8.71 |
0.96 |
12 |
9.00 |
0.96 |
15 |
9.25 |
2.24 |
15 |
9.50 |
1.63 |
13 |
7 |
13.08 |
1.19 |
12 |
13.32 |
1.41 |
15 |
13.82 |
2.99 |
15 |
14.11 |
2.75 |
13 |
14 |
25.84 |
2.44 |
12 |
25.38 |
2.20 |
15 |
26.77 |
5.39 |
15 |
28.48 |
5.22 |
13 |
Key: N = Number of observations
SD = Standard deviation
R = Retained after standardization of litters
Body Weight Gain (g) of Male Pups
Lactation Day |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
0-4 |
3.12 |
0.82 |
12 |
3.18 |
0.64 |
15 |
3.36 |
1.48 |
15 |
3.70 |
1.17 |
12 |
4R-7 |
4.48 |
0.59 |
12 |
4.28 |
0.76 |
15 |
4.65 |
1.18 |
15 |
4.72 |
1.35 |
12 |
7-14 |
12.91 |
2.14 |
12 |
12.12 |
1.35 |
15 |
12.87 |
2.82 |
15 |
14.11 |
2.93 |
12 |
Key: N = Number of observations
SD = Standard deviation
R = Retained after standardization of litters
Body Weight Gain (g) of Female Pups
Lactation Day |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
0-4 |
3.05 |
0.72 |
12 |
3.25 |
0.62 |
15 |
3.38 |
1.54 |
15 |
3.77 |
1.17 |
12 |
4R-7 |
4.31 |
0.65 |
12 |
4.35 |
0.75 |
15 |
4.54 |
1.06 |
15 |
4.47 |
1.27 |
13 |
7-14 |
12.53 |
1.39 |
12 |
12.03 |
1.23 |
15 |
13.06 |
2.78 |
15 |
14.09 |
3.45 |
12 |
Key: N = Number of observations
SD = Standard deviation
R = Retained after standardization of litters
Body Weight Gain (g) of Male and Female Pups
Lactation Day |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
0-4 |
3.08 |
0.75 |
12 |
3.21 |
0.64 |
15 |
3.37 |
1.51 |
15 |
3.69 |
1.10 |
13 |
4R-7 |
4.37 |
0.49 |
12 |
4.31 |
0.75 |
15 |
4.57 |
1.06 |
15 |
4.61 |
1.26 |
13 |
7-14 |
12.76 |
1.74 |
12 |
12.06 |
1.25 |
15 |
12.95 |
2.77 |
15 |
14.37 |
3.15 |
13 |
Key: N = Number of observations
SD = Standard deviation
R = Retained after standardization of litters
Ano-genital Distance (mm) of Male Pups
Lactation Day |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
0 |
1.92 |
0.05 |
12 |
1.93 |
0.04 |
15 |
1.93 |
0.08 |
15 |
1.96 |
0.07 |
13 |
Key: N = Number of observations
SD = Standard deviation
Ano-genital Distance (mm) of Female Pups
Lactation Day |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
0 |
0.81 |
0.04 |
12 |
0.81 |
0.03 |
15 |
0.81 |
0.03 |
15 |
0.82 |
0.05 |
14 |
Key: N = Number of observations
SD = Standard deviation
Summary of Effect on Fertility, Reproduction and Development
Observations |
Values |
||||||
Dose Level(mg/kg bw/day) |
0 |
100 |
500 |
1000 |
|||
Pairs started (N) |
15 |
15 |
15 |
15 |
|||
Females showing evidence of copulation (N) |
14 |
15 |
15 |
15 |
|||
Females achieving pregnancy @ (N) |
12 |
15 |
15 |
15 |
|||
Conceiving days 1 – 5 (N) |
12 |
14 |
14 |
14 |
|||
Conceiving days 6 –10 (N) |
0 |
0 |
1 |
1 |
|||
Conceiving days ≥ 11 days (N) |
1 |
1 |
0 |
0 |
|||
Pregnancy = 21 days (N) |
0 |
0 |
0 |
0 |
|||
Pregnancy = 22 days (N) |
8 |
9 |
7 |
6 |
|||
Pregnancy = 23 days (N) |
2 |
5 |
8 |
8 |
|||
Pregnancy³24 days (N) |
1 |
1 |
0 |
0 |
|||
Female sacrificed on post-coitum day 25 (N) |
3 |
0 |
0 |
1 |
|||
Dams with live pups born (N) |
12 |
15 |
15 |
14 |
|||
Dams with live pups on day 4 (N) |
12 |
15 |
15 |
13 |
|||
Dams with live pups on day 14 (N) |
12 |
15 |
15 |
13 |
|||
N° of female rats sacrificed with live pups on PND 14 |
0 |
0 |
0 |
0 |
|||
N° of female rats with no live pups on PND 14 |
0 |
0 |
0 |
1 |
|||
Mean N° of Implants (Mean±SD) |
13.50 ± 1.51 |
12.47 ± 1.06 |
12.27 ±2.02 |
11.07±4.33 |
|||
ABNORMAL PUPS |
|||||||
Dams with 0 |
10 |
15 |
14 |
13 |
|||
Dams with 1 |
2 |
0 |
0 |
0 |
|||
Dams with >2 |
0 |
0 |
1 |
1 |
|||
LOSS OF OFFSPRING |
|||||||
Pre-natal / Post-implantation (implantations minus live births) |
|||||||
Females with 0 (N) |
6 |
3 |
6 |
3 |
|||
Females with 1 (N) |
2 |
6 |
2 |
5 |
|||
Females with 2 (N) |
3 |
5 |
4 |
1 |
|||
Females with ≥ 3 (N) |
1 |
1 |
3 |
5 |
|||
Post-natal (live births minus alive at post-natal day 4) |
|||||||
Females with 0 |
5 |
10 |
12 |
9 |
|||
Females with 1 |
6 |
3 |
1 |
2 |
|||
Females with 2 |
1 |
0 |
0 |
1 |
|||
Females with ≥ 3 |
0 |
2 |
2 |
2 |
|||
FERTILITY DATA |
|||||||
Number of males housed with females |
15 |
15 |
15 |
15 |
|||
Number of males impregnating females |
12 |
15 |
15 |
15 |
|||
Number of females housed with males |
15 |
15 |
15 |
15 |
|||
Number of females with sperm positive vaginal smear |
14 |
15 |
15 |
15 |
|||
Number of females giving birth |
12 |
15 |
15 |
14 |
|||
No. of Females giving birth to at least one viable pup |
12 |
15 |
15 |
14 |
|||
No. of Females giving birth to all viable pups |
12 |
14 |
14 |
10 |
|||
No. of Females giving birth + Implants |
12 |
15 |
15 |
15 |
|||
No. of Male with confirmed mating |
14 |
15 |
15 |
15 |
|||
No. of Female with Normal Estrous cycle |
15 |
14 |
15 |
15 |
|||
Duration of gestation (day) (mean±SD) |
22.36 ± 0.67 |
22.47 ± 0.64 |
22.53 ± 0.52 |
22.57 ± 0.51 |
|||
Pre-coital interval (day) (mean ± SD) |
3.23 ± 3.49 |
3.20 ± 3.12 |
3.27 ± 1.71 |
2.47 ± 1.64 |
|||
Mean N° of Normal Estrous cycle (mean ± SD) |
2.67 ± 0.49 |
2.60 ± 0.83 |
2.87 ± 0.35 |
2.67 ± 0.49 |
|||
Estrous cycle length (mean ± SD) |
4.00 ± 0.09 |
3.98 ± 0.09 |
4.00 ± 0.13 |
4.09 ± 0.27 |
|||
FERTILITY INDEX |
|||||||
Male Fertility Index (%) |
80.00 |
100.00 |
100.00 |
100.00 |
|||
Female Fertility Index (%) |
80.00 |
100.00 |
100.00 |
100.00 |
|||
Gestation Index (%) |
100.00 |
100.00 |
100.00 |
93.33 |
|||
Parturition Index (%) |
100.00 |
100.00 |
100.00 |
93.33 |
|||
Percentage of Pregnant Rats (%) |
85.71 |
100.00 |
100.00 |
100.00 |
|||
Percentage of Non-pregnant Rats (%) |
14.29 |
0.00 |
0.00 |
0.00 |
|||
Male Mating index (%) |
93.33 |
100.00 |
100.00 |
100.00 |
|||
Female Mating index (%) |
93.33 |
100.00 |
100.00 |
100.00 |
|||
Pre-natal loss (mean±SD) |
1.00±1.28 |
1.27±0.88 |
1.67±2.06 |
2.14±2.03 |
|||
Post-natal loss (mean±SD) |
0.67±0.65 |
0.67±1.23 |
0.53±1.25Φ |
1.43±2.85 |
|||
Pre-natal loss (%) (mean±SD) |
6.96±8.64 |
10.17±7.04 |
13.77±17.53 |
19.18±17.44Φ |
|||
Post-natal loss (%) (mean±SD) |
5.52±5.69 |
5.79±10.38Φ |
4.28±9.38 |
14.43±31.23 |
|||
Key: N = Number
SD = Standard Deviation
N° = Number
% = Percentage
@ = with Implant
Φ= Significantly different from controls (5%),Cochran's test
Note: Rat N° 63, 65, 68 (controls) and 113 (high dose) were sacrificed on gestation day 25.
Females with abnormal oestrus cycle wereexcluded from statistical analysis ofoestrus cycle length.
Presumed pregnant females were excluded from statistical analysis ofpre-coital interval.
Live Birth Index (%) and Postnatal Loss on PND-4 (N°)
Treatment |
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
||||||||
Sex |
M |
F |
M+F |
M |
F |
M+F |
M |
F |
M+F |
M |
F |
M+F |
Live Birth Index (%) |
100.00 |
100.00 |
100.00 |
100.00 |
98.70 |
99.41 |
100.00 |
98.94 |
99.38 |
96.00 |
94.03* |
95.07* |
Postnatal Loss (N°) |
4 |
4 |
8 |
9 |
2 |
11 |
4 |
5 |
9 |
13 |
14 |
27 |
Key:% = Percentage
N° = Number
M = Male
F = Female
*= Significantly different from controls (5%),Chi-square test
Litter Size (Number)
Treatment |
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
||||||||
Sex |
M |
F |
M+F |
M |
F |
M+F |
M |
F |
M+F |
M |
F |
M+F |
Total N° of Pups on PND 0 |
74 |
76 |
150 |
92 |
77 |
169 |
66 |
94 |
160 |
75 |
67 |
142 |
Cannibalised Pups on Day of littering |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Pups Still birth/dead on Day of Littering |
0 |
0 |
0 |
0 |
1 |
1 |
0 |
1 |
1 |
3 |
4 |
7 |
Total N° of Live Pups on PND 0 |
74 |
76 |
150 |
92 |
76 |
168 |
66 |
93 |
159 |
72 |
63 |
135 |
Litter Size on PND 4 |
70 |
72 |
142 |
83 |
75 |
158 |
62 |
89 |
151 |
62 |
53 |
115 |
Litter Size on PND 4 (Retained) |
49 |
47 |
96 |
60 |
60 |
120 |
52 |
63 |
115 |
47 |
40 |
87 |
Litter Size on PND 7 |
48 |
46 |
94 |
59 |
59 |
118 |
49 |
62 |
111 |
44 |
39 |
83 |
Litter Size on PND 14 |
47 |
45 |
92 |
59 |
59 |
118 |
47 |
61 |
108 |
42 |
36 |
78 |
Key:M = Male
F = Female
Pup Mortality and Mortality Index
Treatment |
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
||||||||
Sex |
M |
F |
M+F |
M |
F |
M+F |
M |
F |
M+F |
M |
F |
M+F |
Mortality During PND 0 – 4 (N°) |
4 |
4 |
8 |
9 |
2 |
11 |
4 |
5 |
9 |
13 |
14 |
27 |
Mortality index during PND 0-4 (%) |
5.41 |
5.26 |
5.33 |
9.78 |
2.60 |
6.51 |
6.06 |
5.32 |
5.63 |
17.33 |
20.90* |
19.01* |
Mortality During PND 4R - 7 (N°) |
1 |
1 |
2 |
1 |
1 |
2 |
3 |
1 |
4 |
3 |
1 |
4 |
Mortality index during PND 4R-7 (%) |
2.04 |
2.13 |
2.08 |
1.67 |
1.67 |
1.67 |
5.77 |
1.59 |
3.48 |
6.38 |
2.50 |
4.60 |
Mortality During PND 4R - 14 (N°) |
2 |
2 |
4 |
1 |
1 |
2 |
5 |
2 |
7 |
5 |
4 |
9 |
Mortality index during PND 4R-14 (%) |
4.08 |
4.26 |
4.17 |
1.67 |
1.67 |
1.67 |
9.62 |
3.17 |
6.09 |
10.64 |
10.00 |
10.34 |
Key: PND = Postnatal day
N° = Number
M = Male
F = Female
*= Significantly different from control (5%),Chi-square test
Pup Survival Index (%)
Group |
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
||||||||
Sex |
M |
F |
M+F |
M |
F |
M+F |
M |
F |
M+F |
M |
F |
M+F |
Survival Indexon PND 4 (%) |
94.59 |
94.74 |
94.67 |
90.22 |
97.40 |
93.49 |
93.94 |
94.68 |
94.38 |
82.67 |
79.10* |
80.99* |
Survival Indexon PND 7 (%) |
97.96 |
97.87 |
97.92 |
98.33 |
98.33 |
98.33 |
94.23 |
98.41 |
96.52 |
93.62 |
97.50 |
95.40 |
Survival Indexon PND 14 (%) |
95.92 |
95.74 |
95.83 |
98.33 |
98.33 |
98.33 |
90.38 |
96.83 |
93.91 |
89.36 |
90.00 |
89.66 |
Key: PND = Postnatal day
% = Percent
M = Male
F = Female
*= Significantly different from control (5%),Chi-square test
Organ Weight (g)of Parental Males
Parameters |
Treatment |
|||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||
(N = 15) |
(N = 15) |
(N = 15) |
(N = 15) |
|||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
Terminal Body weight |
417.260 |
16.023 |
421.367 |
20.641 |
408.753 |
22.115 |
403.767 |
21.394 |
Brain |
2.036 |
0.092 |
2.061 |
0.099 |
2.061 |
0.103 |
2.074 |
0.091 |
Testes |
3.718 |
0.869 |
4.049 |
0.231 |
4.043 |
0.319 |
3.941 |
0.598 |
Cowper’s glands |
0.149 |
0.028 |
0.162 |
0.028 |
0.137 |
0.034 |
0.140 |
0.027 |
Glans penis |
0.091 |
0.008 |
0.089 |
0.010 |
0.093 |
0.006 |
0.094 |
0.009 |
LABC |
1.209 |
0.162 |
1.273 |
0.159 |
1.124 |
0.176 |
1.224 |
0.178 |
Epididymis |
1.423 |
0.145 |
1.518 |
0.116 |
1.451 |
0.111 |
1.383 |
0.162 |
Prostate and Seminal vesicles with coagulating gland |
2.432 |
0.299 |
2.474 |
0.313 |
2.351 |
0.271 |
2.401 |
0.355 |
Thyroid gland |
0.0153 |
0.0014 |
0.0152 |
0.0021 |
0.0155 |
0.0023 |
0.0157 |
0.0023 |
Key: N = Number
SD = Standard deviation
Organ Weight (g)of Parental Females
Parameters |
Treatment |
|||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||
(N = 12) |
(N = 15) |
(N = 15) |
(N = 14) |
|||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
Terminal Body Weight |
269.467 |
16.032 |
267.213 |
12.511 |
267.033 |
17.960 |
263.607 |
16.687 |
Brain |
1.971 |
0.136 |
1.954 |
0.099 |
1.941 |
0.133 |
2.003 |
0.089 |
Ovaries |
0.099 |
0.018 |
0.093 |
0.016 |
0.103 |
0.013 |
0.102 |
0.017 |
Uterus including cervix |
0.564 |
0.108 |
0.585 |
0.097 |
0.590 |
0.127 |
0.619 |
0.122 |
Thyroid gland |
0.0137 |
0.0020 |
0.0162€ |
0.0029 |
0.0150 |
0.0021 |
0.0158 |
0.0022 |
Key: N = Number
SD = Standard deviation
€= Significantly different from control (5%),Dunnett's test with Bonferroni adjustment
Note: Organ weight was not recorded of dam sacrificed on GD-25 from control group (damNo63, 65, 68) and high dose group (damNo113).
Organ Weight Relative to Terminal BodyWeight (%)of Parental Males
Parameters |
Treatment |
|||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||
(N = 15) |
(N = 15) |
(N = 15) |
(N = 15) |
|||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
Brain |
0.488 |
0.025 |
0.490 |
0.030 |
0.506 |
0.037 |
0.515 |
0.032 |
Testes |
0.890 |
0.203 |
0.962 |
0.058 |
0.989 |
0.048 |
0.975 |
0.141 |
Cowper’s glands |
0.036 |
0.006 |
0.038 |
0.006 |
0.033 |
0.008 |
0.035 |
0.006 |
Glans penis |
0.022 |
0.002 |
0.021 |
0.003 |
0.023 |
0.001 |
0.023 |
0.003 |
LABC |
0.290 |
0.039 |
0.302 |
0.031 |
0.274 |
0.037 |
0.302 |
0.033 |
Epididymis |
0.341 |
0.032 |
0.360 |
0.025 |
0.355 |
0.024 |
0.342 |
0.037 |
Prostate and Seminal vesicles with coagulating gland |
0.584 |
0.074 |
0.587 |
0.070 |
0.575 |
0.061 |
0.596 |
0.089 |
Thyroid gland |
0.0037 |
0.0003 |
0.0036 |
0.0005 |
0.0038 |
0.0006 |
0.0039 |
0.0005 |
Key: N = Number
SD = Standard deviation
Organ Weight Relative to Terminal BodyWeight (%)of Parental Females
Parameters |
Treatment |
|||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||
(N = 12) |
(N = 15) |
(N = 15) |
(N = 14) |
|||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
Brain |
0.732 |
0.048 |
0.733 |
0.049 |
0.729 |
0.061 |
0.763 |
0.064 |
Ovaries |
0.037 |
0.006 |
0.035 |
0.005 |
0.039 |
0.005 |
0.039 |
0.007 |
Uterus including cervix |
0.209 |
0.035 |
0.219 |
0.033 |
0.221 |
0.044 |
0.236 |
0.052 |
Thyroid gland |
0.0051 |
0.0007 |
0.0060€ |
0.0011 |
0.0056 |
0.0007 |
0.0061€ |
0.0011 |
Key: N = Number
SD = Standard deviation
€= Significantly different from control (5%),Dunnett's test with Bonferroni adjustment
Note: Organ weight was not recorded of dam sacrificed on GD-25 from control group (damNo63, 65, 68) and high dose group (damNo113).
Absolute Organ Weight (g) and Relative Organ Weight (g) of Male Pups(PND 14)
Parameters |
Treatment |
|||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||
(N = 12) |
(N = 15) |
(N = 15) |
(N = 12) |
|||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
Terminal Body Weight |
25.7333 |
4.6482 |
25.0800 |
2.7169 |
26.6800 |
5.6371 |
29.3083 |
5.3638 |
Absolute Organ Weight |
||||||||
Thyroid gland |
0.0058 |
0.0027 |
0.0049 |
0.0010 |
0.0053 |
0.0010 |
0.0055 |
0.0013 |
Relative Organ Weight |
||||||||
Thyroid gland |
0.0226 |
0.0093 |
0.0198 |
0.0046 |
0.0202 |
0.0046 |
0.0188 |
0.0045 |
Key: N = Number
SD = Standard deviation
Absolute Organ Weight (g) and Relative Organ Weight (g) of Female Pups(PND 14)
Parameters |
Treatment |
|||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||
(N = 12) |
(N = 15) |
(N = 15) |
(N = 12) |
|||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
B. wt.(TS) |
25.6167 |
1.9035 |
25.5333 |
2.3666 |
26.6733 |
5.1206 |
27.8333 |
5.6103 |
Absolute Organ Weight |
||||||||
Thyroid gland |
0.0065 |
0.0032 |
0.0050 |
0.0014 |
0.0055 |
0.0009 |
0.0057 |
0.0007 |
Relative Organ Weight |
||||||||
Thyroid gland |
0.0253 |
0.0124 |
0.0201Φ |
0.0064 |
0.0212 |
0.0042 |
0.0207 |
0.0030 |
Key: N = Number
SD = Standard deviation
Φ = Significantly different from control (5%),Cochran's t test
Thyroid Hormone Levels in PND 4 Pups
Hormone |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
T4 |
17.558 |
2.304 |
12 |
16.160 |
2.794 |
13 |
15.029 |
2.634 |
11 |
14.493 *€ |
2.617 |
8 |
Key: N = Number
SD = Standard deviation
* =Significantly different from control (p£0.05)
€= Dunnett's t-Test with Bonferroni adjustment
Thyroid Hormone Levels in PND 14Pups
Hormone |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
T3 |
0.357 |
0.043 |
12 |
0.416 **€ |
0.049 |
15 |
0.417 **€ |
0.064 |
15 |
0.422 **€ |
0.035 |
13 |
T4 |
59.982 |
5.207 |
12 |
66.385 |
6.349 |
15 |
61.141 |
10.534 |
15 |
62.082 |
9.020 |
13 |
TSH |
0.611 |
0.233 |
12 |
0.575 |
0.294 |
15 |
0.844 |
0.738 |
15 |
0.602 |
0.331 |
13 |
Key: N = Number
SD = Standard deviation
**=Significantly different from control (p£0.01)
€= Dunnett's t-Test with Bonferroni adjustment
Thyroid Hormone Levels in Parental Males
Hormone |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
T3 |
0.251 |
0.045 |
15 |
0.229 |
0.045 |
15 |
0.262 |
0.082 |
15 |
0.262 |
0.040 |
15 |
T4 |
61.028 |
6.545 |
15 |
57.778 |
4.664 |
15 |
54.306 *# |
6.956 |
15 |
52.873 **# |
6.957 |
15 |
TSH |
1.042 |
0.602 |
13 |
0.659 |
0.353 |
14 |
0.999 |
0.418 |
15 |
1.385 |
0.702 |
14 |
Key: N = Number
SD = Standard deviation
* =Significantly different from control (p£0.05)
**=Significantly different from control (p£0.01)
#= Dunnett's t-Test
Thyroid Hormone Levels in Parental Females
Hormone |
Treatment |
|||||||||||
0 mg/kg bw/day |
100 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
|||||||||
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
Mean |
SD |
N |
|
T3 |
0.297 |
0.064 |
12 |
0.297 |
0.063 |
15 |
0.287 |
0.072 |
15 |
0.305 |
0.041 |
14 |
T4 |
44.218 |
7.981 |
12 |
41.155 |
5.397 |
15 |
42.930 |
8.303 |
15 |
44.187 |
7.156 |
14 |
TSH |
1.627 |
0.507 |
12 |
1.676 |
0.572 |
15 |
1.579 |
0.626 |
15 |
1.548 |
0.737 |
14 |
Key: N = Number
SD = Standard deviation
Applicant's summary and conclusion
- Conclusions:
- The No Observed Adverse Effect Level (NOAEL) for systemic toxicity of parental rats was found to be 1000 mg/kg/day as no treatment related adverse effects were observed.
The reproductive and fertility NOAEL of parental rats was 1000 mg/kg/day as no treatment-related effects were observed on reproduction and fertility endpoints. The developmental NOAEL of F1 pups was 500 mg/kg/day, based on adverse effects observed on pre-natal loss and live birth index at a dose level of 1000 mg/kg/day. - Executive summary:
Potential reproductive toxicity has been investigated in the rat in a GLP compliant screening study conducted according to OECD TG 421. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity and reproductive toxicity and fertility
in parental animals was 1000 mg/kg bw/day. The NOAEL for developmental toxicity of pups was 500 mg/kg/day.
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